The long-term goal of the proposal is to decipher the function of mitochondrial orphan enzymes and their regulations on unexplored metabolic pathways with potential implications for human diseases. Mitochondria are intracellular organelles that house not only the machinery for ATP synthesis and fuel oxidation, but also metabolic enzymes for synthesis of building blocks for cell growth. For instance, a critical mitochondrial enzyme methylmalonyl-CoA mutase (MUT) recycles branched carbon chains from amino acids and lipids into tricarboxylic acid cycle. This process is critically dependent on an essential coenzyme vitamin B12 (B12). This project focuses on the role played by CLYBL, a ubiquitously expressed mitochondrial enzyme of unknown function, in regulating mitochondrial B12 function. A loss-of-function variant of CLYBL has been linked to subclinical B12 deficiency by two recent human genome-wide association studies. This proposal builds on the computational genomics analysis performed in our laboratory, which suggested CLYBL lies at the heart of a novel mitochondrial metabolic pathway connecting to B12-dependent MUT function. The proposed research seeks to understand: (1) enzymatic activities of CLYBL; (2) the CLYBL-dependent regulation of mitochondrial B12 function; and (3) molecular mechanism underlying this regulation. These studies will take advantage of the laboratory's expertise in advanced mass spectrometry-based metabolomics, computational genomics and mitochondrial physiology, combined with my skills in biochemistry and cell biology, to decipher the physiological function of CLYBL. This study of CLYBL represents a great example of reverse human genetics. This work promises to shed new insights on mitochondrial biochemistry with potential implications for human diseases.